Cataract is the leading cause of blindness worldwide and its only cure, surgical removal, can result in complications and places an enormous financial burden on our economy. The prevalence of cataract in the U.S. is expected to more than double by the year 2050. Despite decades of research, the causes of age-related protein aggregation and lens opacity remain ill- defined and no therapies exist to prevent or delay cataract. The long-term goals of our research are to identify modifications to lens proteins during aging and cataractogenesis in order to understand protein aging mechanisms and to develop ways to prevent or delay deleterious events. Recently, we have discovered an age-dependent, non-enzymatic biochemical mechanism that results in significant amounts of both age-related lens protein modification and cataract-related lens protein crosslinking. This chemistry occurs at phosphoserine and cysteine residues and involves modification by glutathione (GSH) and other nucleophiles in the lens including protein thiols resulting in irreversible protein crosslinking. Or hypothesis is that lens protein non-disulfide thiolation by GSH prevents protein-protein crosslinking, but with age this protective mechanism is overwhelmed resulting in irreversible crosslinking, protein aggregation, and lens opacification. To test this hypothesis we will employ state-of-the-art proteomics methodology to further define age-related and cataract-specific modifications, evaluate conditions upon which the chemistry occurs, and validate an animal model for detected modifications and future therapeutic use. Specifically we propose to: 1) characterize and quantify protein non-disulfide thiol modifications and protein crosslinks in normal and cataractous human lenses, 2) determine conditions controlling non-disulfide thiol modification and protein crosslinking, and 3) characterize lens protein non-disulfide modifications and protein crosslinks in an animal model of human nuclear cataract. The proposed experiments are expected to provide new mechanistic details on protein aging that will not only inform the development of new cataract treatments, but also guide therapeutic development for other aging and protein aggregation diseases.